Streptococcus pneumoniae polynucleotides which encode folyl-polyglutamate synthetase (FPGS) polypeptides

ABSTRACT

The invention provides folC polypeptides and DNA (RNA) encoding folC polypeptides and methods for producing such polypeptides by recombinant techniques. Also provided are methods for utilizing folC polypeptides to screen for antibacterial compounds.

RELATED APPLICATIONS

This application claims benefit of US Provisional Patent Application,60/058024, filed Aug. 29, 1997.

FIELD OF THE INVENTION

This invention relates to newly identified polynucleotides andpolypeptides, and their production and uses, as well as their variants,agonists and antagonists, and their uses. In particular, in these and inother regards, the invention relates to novel polynucleotides andpolypeptides of the fpgs family, hereinafter referred to as "folC".

BACKGROUND OF THE INVENTION

The Streptococci make up a medically important genera of microbes knownto cause several types of disease in humans, including, for example,otitis media, conjunctivitis, pneumonia, bacteremia, meningitis,sinusitis, pleural empyema and endocarditis, and most particularlymeningitis, such as for example infection of cerebrospinal fluid. Sinceits isolation more than 100 years ago, Streptococcus pneumoniae has beenone of the more intensively studied microbes. For example, much of ourearly understanding that DNA is, in fact the genetic material waspredicated on the work of Griffith and of Avery, Macleod and McCartyusing this microbe. Despite the vast amount of research with S.pneumoniae, many questions concerning the virulence of this microberemain. It is particularly preferred to employ Streptococcal genes andgene products as targets for the development of antibiotics.

The frequency of Streptococcus pneumoniae infections has risendramatically in the past 20 years. This has been attributed to theemergence of multiply antibiotic resistant strains and an increasingpopulation of people with weakened immune systems. It is no longeruncommon to isolate Streptococcus pneumoniae strains which are resistantto some or all of the standard antibiotics. This has created a demandfor both new anti-microbial agents and diagnostic tests for thisorganism.

Folate derivatives are essential cofactors in the biosynthesis ofpurines, pyrimidines and amino acids in living cells. Whereas animalsrequire folate in their diet, most bacterial species synthesize theirown and are thus subject to inhibition by compounds that inhibit thefolate synthesis pathway (see, Lacks etal, 1995:J.Bacteriology 1:66-74).The sulphonamides and trimethoprim are two examples of antibiotics thateffectively block folate biosynthesis. FolC of E.coli, has both bothdihydrofolate synthetase (DHFS) and folyl-polyglutamate synthetase(FPGS) activity. FPGS of S.pneumoniae is necessary for a fullyfunctional folate synthesis pathway.

Clearly, there is a need for factors, such as the novel compounds of theinvention, that have a present benefit of being useful to screencompounds for antibiotic activity. Such factors are also useful todetermine their role in pathogenesis of infection, dysfunction anddisease. There is also a need for identification and characterization ofsuch factors and their antagonists and agonists which can play a role inpreventing, ameliorating or correcting infections, dysfunctions ordiseases.

The polypeptides of the invention have amino acid sequence homology to aknown sulB protein.

SUMMARY OF THE INVENTION

It is an object of the invention to provide polypeptides that have beenidentified as novel folC polypeptides by homology between the amino acidsequence set out in Table 1 SEQ ID NO: 2! and a known amino acidsequence or sequences of other proteins such as sulB protein.

It is a further object of the invention to provide polynucleotides thatencode folC polypeptides, particularly polynucleotides that encode thepolypeptide herein designated folC.

In a particularly preferred embodiment of the invention thepolynucleotide comprises a region encoding folC polypeptides comprisingthe sequence set out in Table 1 SEQ ID NO:1! which includes a fulllength gene, or a variant thereof.

In another particularly preferred embodiment of the invention there is anovel folC protein from Streptococcus pneumoniae comprising the aminoacid sequence of Table 1 SEQ ID NO:2!, or a variant thereof.

In accordance with another aspect of the invention there is provided anisolated nucleic acid molecule encoding a mature polypeptide expressibleby the Streptococcus pneumoniae 0100993 strain contained in thedeposited strain.

A further aspect of the invention there are provided isolated nucleicacid molecules encoding folC, particularly Streptococcus pneumoniaefolC, including mRNAs, cDNAs, genomic DNAs. Further embodiments of theinvention include biologically, diagnostically, prophylactically,clinically or therapeutically useful variants thereof, and compositionscomprising the same.

In accordance with another aspect of the invention, there is providedthe use of a polynucleotide of the invention for therapeutic orprophylactic purposes, in particular genetic immunization. Among theparticularly preferred embodiments of the invention are naturallyoccurring allelic variants of folC and polypeptides encoded thereby.

Another aspect of the invention there are provided novel polypeptides ofStreptococcus pneumoniae referred to herein as folC as well asbiologically, diagnostically, prophylactically, clinically ortherapeutically useful variants thereof, and compositions comprising thesame.

Among the particularly preferred embodiments of the invention arevariants of folC polypeptide encoded by naturally occurring alleles ofthe folC gene.

In a preferred embodiment of the invention there are provided methodsfor producing the aforementioned folC polypeptides.

In accordance with yet another aspect of the invention, there areprovided inhibitors to such polypeptides, useful as antibacterialagents, including, for example, antibodies.

In accordance with certain preferred embodiments of the invention, thereare provided products, compositions and methods for assessing folCexpression, treating disease, for example, otitis media, conjunctivitis,pneumonia, bacteremia, meningitis, sinusitis, pleural empyema andendocarditis, and most particularly meningitis, such as for exampleinfection of cerebrospinal fluid, assaying genetic variation, andadministering a folC polypeptide or polynucleotide to an organism toraise an immunological response against a bacteria, especially aStreptococcus pneumoniae bacteria.

In accordance with certain preferred embodiments of this and otheraspects of the invention there are provided polynucleotides thathybridize to folC polynucleotide sequences, particularly under stringentconditions.

In certain preferred embodiments of the invention there are providedantibodies against folC polypeptides.

In other embodiments of the invention there are provided methods foridentifying compounds which bind to or otherwise interact with andinhibit or activate an activity of a polypeptide or polynucleotide ofthe invention comprising: contacting a polypeptide or polynucleotide ofthe invention with a compound to be screened under conditions to permitbinding to or other interaction between the compound and the polypeptideor polynucleotide to assess the binding to or other interaction with thecompound, such binding or interaction being associated with a secondcomponent capable of providing a detectable signal in response to thebinding or interaction of the polypeptide or polynucleotide with thecompound; and determining whether the compound binds to or otherwiseinteracts with and activates or inhibits an activity of the polypeptideor polynucleotide by detecting the presence or absence of a signalgenerated from the binding or interaction of the compound with thepolypeptide or polynucleotide.

In accordance with yet another aspect of the invention, there areprovided folC agonists and antagonists, preferably bacteriostatic orbacteriocidal agonists and antagonists.

In a further aspect of the invention there are provided compositionscomprising a folC polynucleotide or a folC polypeptide foradministration to a cell or to a multicellular organism.

Various changes and modifications within the spirit and scope of thedisclosed invention will become readily apparent to those skilled in theart from reading the following descriptions and from reading the otherparts of the present disclosure.

GLOSSARY

The following definitions are provided to facilitate understanding ofcertain terms used frequently herein.

"Host cell" is a cell which has been transformed or transfected, or iscapable of transformation or transfection by an exogenous polynucleotidesequence.

"Identity," as known in the art, is a relationship between two or morepolypeptide sequences or two or more polynucleotide sequences, asdetermined by comparing the sequences. In the art, "identity" also meansthe degree of sequence relatedness between polypeptide or polynucleotidesequences, as the case may be, as determined by the match betweenstrings of such sequences. "Identity" and "similarity" can be readilycalculated by known methods, including but not limited to thosedescribed in (Computational Molecular Biology, Lesk, A. M., ed., OxfordUniversity Press, New York, 1988; Biocomputing: Informatics and GenomeProjects, Smith, D. W., ed., Academic Press, New York, 1993; ComputerAnalysis of Sequence Data, Part I, Griffin, A. M., and Griffin, H. G.,eds., Humana Press, New Jersey, 1994; Sequence Analysis in MolecularBiology, von Heinje, G., Academic Press, 1987; and Sequence AnalysisPrimer, Gribskov, M. and Devereux, J., eds., M Stockton Press, New York,1991; and Carillo, H., and Lipman, D., SIAM J. Applied Math., 48: 1073(1988). Preferred methods to determine identity are designed to give thelargest match between the sequences tested. Methods to determineidentity and similarity are codified in publicly available computerprograms. Preferred computer program methods to determine identity andsimilarity between two sequences include, but are not limited to, theGCG program package (Devereux, J., et al., Nucleic Acids Research 12(1):387 (1984)), BLASTP, BLASTN, and FASTA (Atschul, S. F. et al., J. Molec.Biol. 215: 403-410 (1990). The BLAST X program is publicly availablefrom NCBI and other sources (BLAST Manual, Altschul, S., et al., NCBINLM NIH Bethesda, Md. 20894; Altschul, S., et al., J. Mol. Biol. 215:403-410 (1990). As an illustration, by a polynucleotide having anucleotide sequence having at least, for example, 95% "identity" to areference nucleotide sequence of SEQ ID NO:1 it is intended that thenucleotide sequence of the polynucleotide is identical to the referencesequence except that the polynucleotide sequence may include up to fivepoint mutations per each 100 nucleotides of the reference nucleotidesequence of SEQ ID NO:1. In other words, to obtain a polynucleotidehaving a nucleotide sequence at least 95% identical to a referencenucleotide sequence, up to 5% of the nucleotides in the referencesequence may be deleted or substituted with another nucleotide, or anumber of nucleotides up to 5% of the total nucleotides in the referencesequence may be inserted into the reference sequence. These mutations ofthe reference sequence may occur at the 5 or 3 terminal positions of thereference nucleotide sequence or anywhere between those terminalpositions, interspersed either individually among nucleotides in thereference sequence or in one or more contiguous groups within thereference sequence. Analogously , by a polypeptide having an amino acidsequence having at least, for example, 95% identity to a reference aminoacid sequence of SEQ ID NO:2 is intended that the amino acid sequence ofthe polypeptide is identical to the reference sequence except that thepolypeptide sequence may include up to five amino acid alterations pereach 100 amino acids of the reference amino acid of SEQ ID NO:2. Inother words, to obtain a polypeptide having an amino acid sequence atleast 95% identical to a reference amino acid sequence, up to 5% of theamino acid residues in the reference sequence may be deleted orsubstituted with another amino acid, or a number of amino acids up to 5%of the total amino acid residues in the reference sequence may beinserted into the reference sequence. These alterations of the referencesequence may occur at the amino or carboxy terminal positions of thereference amino acid sequence or anywhere between those terminalpositions, interspersed either individually among residues in thereference sequence or in one or more contiguous groups within thereference sequence.

"Isolated" means altered "by the hand of man" from its natural state,ie., if it occurs in nature, it has been changed or removed from itsoriginal environment, or both. For example, a polynucleotide or apolypeptide naturally present in a living organism is not "isolated,"but the same polynucleotide or polypeptide separated from the coexistingmaterials of its natural state is "isolated", as the term is employedherein.

"Polynucleotide(s)" generally refers to any polyribonucleotide orpolydeoxribonucleotide, which may be unmodified RNA or DNA or modifiedRNA or DNA. "Polynucleotide(s)" include, without limitation, single- anddouble-stranded DNA, DNA that is a mixture of single- anddouble-stranded regions or single-, double- and triple-stranded regions,single- and double-stranded RNA, and RNA that is mixture of single- anddouble-stranded regions, hybrid molecules comprising DNA and RNA thatmay be single-stranded or, more typically, double-stranded, ortriple-stranded regions, or a mixture of single- and double-strandedregions. In addition, "polynucleotide" as used herein refers totriple-stranded regions comprising RNA or DNA or both RNA and DNA. Thestrands in such regions may be from the same molecule or from differentmolecules. The regions may include all of one or more of the molecules,but more typically involve only a region of some of the molecules. Oneof the molecules of a triple-helical region often is an oligonucleotide.As used herein, the term "polynucleotide(s)" also includes DNAs or RNAsas described above that contain one or more modified bases. Thus, DNAsor RNAs with backbones modified for stability or for other reasons are"polynucleotide(s)" as that term is intended herein. Moreover, DNAs orRNAs comprising unusual bases, such as inosine, or modified bases, suchas tritylated bases, to name just two examples, are polynucleotides asthe term is used herein. It will be appreciated that a great variety ofmodifications have been made to DNA and RNA that serve many usefulpurposes known to those of skill in the art. The term"polynucleotide(s)" as it is employed herein embraces such chemically,enzymatically or metabolically modified forms of polynucleotides, aswell as the chemical forms of DNA and RNA characteristic of viruses andcells, including, for example, simple and complex cells."Polynucleotide(s)" also embraces short polynucleotides often referredto as oligonucleotide(s).

"Polypeptide(s)" refers to any peptide or protein comprising two or moreamino acids joined to each other by peptide bonds or modified peptidebonds. "Polypeptide(s)" refers to both short chains, commonly referredto as peptides, oligopeptides and oligomers and to longer chainsgenerally referred to as proteins. Polypeptides may contain amino acidsother than the 20 gene encoded amino acids. "Polypeptide(s)" includethose modified either by natural processes, such as processing and otherpost-translational modifications, but also by chemical modificationtechniques. Such modifications are well described in basic texts and inmore detailed monographs, as well as in a voluminous researchliterature, and they are well known to those of skill in the art. Itwill be appreciated that the same type of modification may be present inthe same or varying degree at several sites in a given polypeptide.Also, a given polypeptide may contain many types of modifications.Modifications can occur anywhere in a polypeptide, including the peptidebackbone, the amino acid side-chains, and the amino or carboxyl termini.Modifications include, for example, acetylation, acylation,ADP-ribosylation, amidation, covalent attachment of flavin, covalentattachment of a heme moiety, covalent attachment of a nucleotide ornucleotide derivative, covalent attachment of a lipid or lipidderivative, covalent attachment of phosphotidylinositol, cross-linking,cyclization, disulfide bond formation, demethylation, formation ofcovalent cross-links, formation of cysteine, formation of pyroglutamate,formylation, gamma-carboxylation, glycosylation, GPI anchor formation,hydroxylation, iodination, methylation, myristoylation, oxidation,proteolytic processing, phosphorylation, prenylation, racemization,glycosylation, lipid attachment, sulfation, gamma-carboxylation ofglutamic acid residues, hydroxylation and ADP-ribosylation,selenoylation, sulfation, transfer-RNA mediated addition of amino acidsto proteins, such as arginylation, and ubiquitination. See, forinstance, PROTEINS--STRUCTURE AND MOLECULAR PROPERTIES, 2nd Ed., T. E.Creighton, W. H. Freeman and Company, New York (1993) and Wold, F.,Posttranslational Protein Modifications: Perspectives and Prospects,pgs. 1-12 in POSTTRANSLATIONAL COVALENT MODIFICATION OF PROTEINS, B. C.Johnson, Ed., Academic Press, New York (1983); Seifter et al., Meth.Enzymol. 182:626-646 (1990) and Rattan et al., Protein Synthesis:Posttranslational Modifications and Aging, Ann. N.Y. Acad. Sci. 663:48-62 (1992). Polypeptides may be branched or cyclic, with or withoutbranching. Cyclic, branched and branched circular polypeptides mayresult from post-translational natural processes and may be made byentirely synthetic methods, as well.

"Variant(s)" as the term is used herein, is a polynucleotide orpolypeptide that differs from a reference polynucleotide or polypeptiderespectively, but retains essential properties. A typical variant of apolynucleotide differs in nucleotide sequence from another, referencepolynucleotide. Changes in the nucleotide sequence of the variant may ormay not alter the amino acid sequence of a polypeptide encoded by thereference polynucleotide. Nucleotide changes may result in amino acidsubstitutions, additions, deletions, fusions and truncations in thepolypeptide encoded by the reference sequence, as discussed below. Atypical variant of a polypeptide differs in amino acid sequence fromanother, reference polypeptide. Generally, differences are limited sothat the sequences of the reference polypeptide and the variant areclosely similar overall and, in many regions, identical. A variant andreference polypeptide may differ in amino acid sequence by one or moresubstitutions, additions, deletions in any combination. A substituted orinserted amino acid residue may or may not be one encoded by the geneticcode. A variant of a polynucleotide or polypeptide may be a naturallyoccurring such as an allelic variant, or it may be a variant that is notknown to occur naturally. Non-naturally occurring variants ofpolynucleotides and polypeptides may be made by mutagenesis techniques,by direct synthesis, and by other recombinant methods known to skilledartisans.

DESCRIPTION OF TH INVENTION

The invention relates to novel folC polypeptides and polynucleotides asdescribed in greater detail below. In particular, the invention relatesto polypeptides and polynucleotides of a novel folC of Streptococcuspneumoniae, which is related by amino acid sequence homology to sulBpolypeptide. The invention relates especially to folC having thenucleotide and amino acid sequences set out in Table 1 SEQ ID NO:1! andTable 1 SEQ ID NO:2! respectively, and to the folC nucleotide sequencesof the DNA in the deposited strain and amino acid sequences encodedthereby.

                                      TABLE 1    __________________________________________________________________________    folC Polynucleotide and Polypeptide Sequences    __________________________________________________________________________    (A) Sequences from Streptococcus pneumoniae folC polynucleotide sequence     SEQ ID NO:1!.    5'-1 ATGTGTATGT                 TTGAAGTAGA                         AGAATGCTTT                                 CATAGTCGGA                                         TTGGTTTGAA    51   TTTTCGATCA                 GGTTTGGCCG                         GAATACAGCA                                 AGCGGTGGAT                                         TTGTTAGGAA    101  ATCCTGAGCA                 GTCTTACCCT                         ATTATCCACG                                 TAACAGGGAC                                         TAATGGGAAA    151  GGATCTACCA                 TTGCTTTTAT                         GAGGGAATTA                                 TTTATGGGGC                                         ATGGCAAAAA    201  AGTTGCGACC                 TTTACCTCCC                         CTCATATCGT                                 ATCCATCAAT                                         GACCGAATTT    251  GCATTAATGG                 GCAACCTATA                         GCAGACGCAG                                 ACTTTATCCG                                         TTTGGCTGAT    301  CAGGTCAAGG                 AGATGGAGAA                         AACGCTTCTG                                 CAAACTCCTG                                         ACCAGTTGTC    351  CTTTTTTGAA                 TTGCTGACCT                         TGGTTGCTTT                                 TCTTTATTTT                                         AGGGAGCAGG    401  AGGTGGATTT                 GGTTTTATTA                         GAAGTGGGAA                                 TTGGTGGCTT                                         ACTTGACACG    451  ACCAATGTGG                 TAACTGGAGA                         GCTTGCTGTC                                 ATCACCTCCA                                         TTGGGCTTGA    501  CCATCAAGAA                 ACCTTGGGTG                         ATAGTCTAGA                                 AGCAATTGCA                                         GAGCAGAAAG    551  CTGGTATTTT                 CAAGGCTGGT                         AAGAAGGCAG                                 TGATTGCGAA                                         ATTGCCTCCA    601  GAAGCTAGGC                 TTGTCTGTCA                         GAAAAAAGCC                                 GAATCTTTAG                                         CTGTTAACCT    651  TTATCAGGCA                 GGTCAAGATT                         TTTTAATGCT                                 GAATGGTGAT                                         TTTTCAAGCT    701  CTTTACTAAA                 TCTTTCACAG                         CTGAACATAG                                 GCTTAGAAGG                                         AGTCTATCAG    751  CAGGAGAATG                 CAGCCTTGGC                         GTTGCAAACT                                 TTTCTTCTTT                                         TTATGGGAGA    801  AGGAAAGGAA                 GCTGTTGATG                         AACAGGCTGT                                 AAGAAAGGCC                                         TTGGAACAGA    851  CCCATTGGGC                 TGGTCGCTTG                         GAGCGTATTC                                 GCCCACAGAT                                         TTACTTGGAT    901  GGTGCTCATA                 ACCTCCCTGC                         CTTGACTCGC                                 TTGGTTGAGT                                         TTATCAAAGA    951  AAAAGAGCAG                 GAAGGTTATC                         GTCCTCAAAT                                 CCTTTTTGGA                                         GCCTTGAAAC    1001 GGAAGGATTA                 TCAAGGGATG                         TTAGGTTACC                                 TGACTGAAAA                                         ATTGCCTCAG    1051 GTGGAACTCA                 AGGTGACCGG                         CTTTGACTAT                                 CAGGGGGCTT                                         TGGACGAAAG    1101 GGATGTGACA                 GGTTACGATA                         TAGTTTCTTC                                 TTACCGAGAA                                         TTTATCAGCG    1151 ATTTTGAAGA                 AAGAGCAGAT                         GCTCAGGATT                                 TGCTGTTCGT                                         TACAGGGTCT    1201 CTCTATTTTA                 TCTCAGAAGT                         ACGGGGCTAC                                 CTGCTGGACC                                         GTGAGCAGAT    1251 AAAT -3'    (B) folC polypeptide sequence deduced from the polynucleotide sequence in    this table  SEQ ID NO:2!.    NH.sub.2 -1         MCMFEVEECF                 HSRIGLNFRS                         GLAGIQQAVD                                 LLGNPEQSYP                                         IIHVTGTNGK    51   GSTIAFMREL                 FMGHGKKVAT                         FTSPHIVSIN                                 DRICINGQPI                                         ADADFIRLAD    101  QVKEMEKTLL                 QTPDQLSFFE                         LLTLVAFLYF                                 REQEVDLVLL                                         EVGIGGLLDT    151  TNVVTGELAV                 ITSIGLDHQE                         TLGDSLEAIA                                 EQKAGIFKAG                                         KKAVIAKLPP    201  EARLVCQKKA                 ESLAVNLYQA                         GQDFLMLNGD                                 FSSSLLNLSQ                                         LNIGLEGVYQ    251  QENAALALQT                 FLLFMGEGKE                         AVDEQAVRKA                                 LEQTHWAGRL                                         ERIRPQIYLD    301  GAHNLPALTR                 LVEFIKEKEQ                         EGYRPQILFG                                 ALKRKDYQGM                                         LGYLTEKLPQ    351  VELKVTGFDY                 QGALDERDVT                         GYDIVSSYRE                                 FISDFEERAD                                         AQDLLFVTGS    401  LYFISEVRGY                 LLDREQIN -COOH    (C) Polynucleotide sequence embodiments  SEQ ID NO: 1!.    X-(R.sub.1).sub.n -1         ATGTGTATGT                 TTGAAGTAGA                         AGAATGCTTT                                 CATAGTCGGA                                         TTGGTTTGAA    51   TTTTCGATCA                 GGTTTGGCCG                         GAATACAGCA                                 AGCGGTGGAT                                         TTGTTAGGAA    101  ATCCTGAGCA                 GTCTTACCCT                         ATTATCCACG                                 TAACAGGGAC                                         TAATGGGAAA    151  GGATCTACCA                 TTGCTTTTAT                         GAGGGAATTA                                 TTTATGGGGC                                         ATGGCAAAAA    201  AGTTGCGACC                 TTTACCTCCC                         CTCATATCGT                                 ATCCATCAAT                                         GACCGAATTT    251  GCATTAATGG                 GCAACCTATA                         GCAGACGCAG                                 ACTTTATCCG                                         TTTGGCTGAT    301  CAGGTCAAGG                 AGATGGAGAA                         AACGCTTCTG                                 CAAACTCCTG                                         ACCAGTTGTC    351  CTTTTTTGAA                 TTGCTGACCT                         TGGTTGCTTT                                 TCTTTATTTT                                         AGGGAGCAGG    401  AGGTGGATTT                 GGTTTTATTA                         GAAGTGGGAA                                 TTGGTGGCTT                                         ACTTGACACG    451  ACCAATGTGG                 TAACTGGAGA                         GCTTGCTGTC                                 ATCACCTCCA                                         TTGGGCTTGA    501  CCATCAAGAA                 ACCTTGGGTG                         ATAGTCTAGA                                 AGCAATTGCA                                         GAGCAGAAAG    551  CTGGTATTTT                 CAAGGCTGGT                         AAGAAGGCAG                                 TGATTGCGAA                                         ATTGCCTCCA    601  GAAGCTAGGC                 TTGTCTGTCA                         GAAAAAAGCC                                 GAATCTTTAG                                         CTGTTAACCT    651  TTATCAGGCA                 GGTCAAGATT                         TTTTAATGCT                                 GAATGGTGAT                                         TTTTCAAGCT    701  CTTTACTAAA                 TCTTTCACAG                         CTGAACATAG                                 GCTTAGAAGG                                         AGTCTATCAG    751  CAGGAGAATG                 CAGCCTTGGC                         GTTGCAAACT                                 TTTCTTCTTT                                         TTATGGGAGA    801  AGGAAAGGAA                 GCTGTTGATG                         AACAGGCTGT                                 AAGAAAGGCC                                         TTGGAACAGA    851  CCCATTGGGC                 TGGTCGCTTG                         GAGCGTATTC                                 GCCCACAGAT                                         TTACTTGGAT    901  GGTGCTCATA                 ACCTCCCTGC                         CTTGACTCGC                                 TTGGTTGAGT                                         TTATCAAAGA    951  AAAAGAGCAG                 GAAGGTTATC                         GTCCTCAAAT                                 CCTTTTTGGA                                         GCCTTGAAAC    1001 GGAAGGATTA                 TCAAGGGATG                         TTAGGTTACC                                 TGACTGAAAA                                         ATTGCCTCAG    1051 GTGGAACTCA                 AGGTGACCGG                         CTTTGACTAT                                 CAGGGGGCTT                                         TGGACGAAAG    1101 GGATGTGACA                 GGTTACGATA                         TAGTTTCTTC                                 TTACCGAGAA                                         TTTATCAGCG    1151 ATTTTGAAGA                 AAGAGCAGAT                         GCTCAGGATT                                 TGCTGTTCGT                                         TACAGGGTCT    1201 CTCTATTTTA                 TCTCAGAAGT                         ACGGGGCTAC                                 CTGCTGGACC                                         GTGAGCAGAT    1251 AAAT -(R.sub.2).sub.n -Y    (D) Polypeptide sequence embodiments  SEQ ID NO:2!.    X-(R.sub.1).sub.n -1         MCMFEVEECF                 HSRIGLNFRS                         GLAGIQQAVD                                 LLGNPEQSYP                                         IIHVTGTNGK    51   GSTIAFMREL                 FMGHGKKVAT                         FTSPHIVSIN                                 DRICINGQPI                                         ADADFIRLAD    101  QVKEMEKTLL                 QTPDQLSFFE                         LLTLVAFLYF                                 REQEVDLVLL                                         EVGIGGLLDT    151  TNVVTGELAV                 ITSIGLDHQE                         TLGDSLEAIA                                 EQKAGIFKAG                                         KKAVIAKLPP    201  EARLVCQKKA                 ESLAVNLYQA                         GQDFLMLNGD                                 FSSSLLNLSQ                                         LNIGLEGVYQ    251  QENAALALQT                 FLLFMGEGKE                         AVDEQAVRKA                                 LEQTHWAGRL                                         ERIRPQIYLD    301  GAHNLPALTR                 LVEFIKEKEQ                         EGYRPQILFG                                 ALKRKDYQGM                                         LGYLTEKLPQ    351  VELKVTGFDY                 QGALDERDVT                         GYDIVSSYRE                                 FISDFEEKAD                                         AQDLLFVTGS    401  LYFISEVRGY                 LLDREQIN -(R.sub.2).sub.n -Y    (E) Sequences from Streptococcus pneumoniae folC polynucleotide ORF    sequence  SEQ ID NO:3!.    5'-1 ATGTGTATGT                 TTGAAGTAGA                         AGAATGCTTT                                 CATAGTCGGA                                         TTGGTTTGAA    51   TTTTCGATCA                 GGTTTGGCCG                         GAATACAGCA                                 AGCGGTGGAT                                         TTGTTAGGAA    101  ATCCTGAGCA                 GTCTTACCCT                         ATTATCCACG                                 TAACAGGGAC                                         TAATGGGAAA    151  GGATCTACCA                 TTGCTTTTAT                         GAGGGAATTA                                 TTTATGGGGC                                         ATGGCAAAAA    201  AGTTGCGACC                 TTTACCTCCC                         CTCATATCGT                                 ATCCATCAAT                                         GACCGAATTT    251  GCATTAATGG                 GCAACCTATA                         GCAGACGCAG                                 ACTTTATCCG                                         TTTGGCTGAT    301  CAGGTCAAGG                 AGATGGAGAA                         AACGCTTCTG                                 CAAACTCCTG                                         ACCAGTTGTC    351  CTTTTTTGAA                 TTGCTGACCT                         TGGTTGCTTT                                 TCTTTATTTT                                         AGGGAGCAGG    401  AGGTGGATTT                 GGTTTTATTA                         GAAGTGGGAA                                 TTGGTGGCTT                                         ACTTGACACG    451  ACCAATGTGG                 TAACTGGAGA                         GCTTGCTGTC                                 ATCACCTCCA                                         TTGGGCTTGA    501  CCATCAAGAA                 ACCTTGGGTG                         ATAGTCTAGA                                 AGCAATTGCA                                         GAGCAGAAAG    551  CTGGTATTTT                 CAAGGCTGGT                         AAGAAGGCAG                                 TGATTGCGAA                                         ATTGCCTCCA    601  GAAGCTAGGC                 TTGTCTGTCA                         GAAAAAAGCC                                 GAATCTTTAG                                         CTGTTAACCT    651  TTATCAGGCA                 GGTCAAGATT                         TTTTAATGCT                                 GAATGGTGAT                                         TTTTCAAGCT    701  CTTTACTAAA                 TCTTTCACAG                         CTGAACATAG                                 GCTTAGAAGG                                         AGTCTATCAG    751  CAGGAGAATG                 CAGCCTTGGC                         GTTGCAAACT                                 TTTCTTCTTT                                         TTATGGGAGA    801  AGGAAAGGAA                 GCTGTTGATG                         AACAGGCTGT                                 AAGAAAGGCC                                         TTGGAACAGA    851  CCCATTGGGC                 TGGTCGCTTG                         GAGCGTATTC                                 GCCCACAGAT                                         TTACTTGGAT    901  GGTGCTCATA                 ACCTCCCTGC                         CTTGACTCGC                                 TTGGTTGAGT                                         TTATCAAAGA    951  AAAAGAGCAG                 GAAGGTTATC                         GTCCTCAAAT                                 CCTTTTTGGA                                         GCCTTGAAAC    1001 GGAAGGATTA                 TCAAGGGATG                         TTAGGTTACC                                 TGACTGAAAA                                         ATTGCCTCAG    1051 GTGGAACTCA                 AGGTGACCGG                         CTTTGACTAT                                 CAGGGGGCTT                                         TGGACGAAAG    1101 GGATGTGACA                 GGTTACGATA                         TAGTTTCTTC                                 TTACCGAGAA                                         TTTATCAGCG    1151 ATTTTGAAGA                 AAGAGCAGAT                         GCTCAGGATT                                 TGCTGTTCGT                                         TACAGGGTCT    1201 CTCTATTTTA                 TCTCAGAAGT                         A-31    (F) folC polypeptide sequence deduced from the polynucleotide ORF    sequence in this table  SEQ ID NO:4!.    NH.sub.2 -1         MCMFEVEECF                 HSRIGLNFRS                         GLAGIQQAVD                                 LLGNPEQSYP                                         IIHVTGTNGK    51   GSTIAFMREL                 FMGHGKKVAT                         FTSPHIVSIN                                 DRICINGQPI                                         ADADFIRLAD    101  QVKEMEKTLL                 QTPDQLSFFE                         LLTLVAFLYF                                 REQEVDLVLL                                         EVGIGGLLDT    151  TNVVTGELAV                 ITSIGLDHQE                         TLGDSLEAIA                                 EQKAGIFKAG                                         KKAVIAKLPP    201  EARLVCQKKA                 ESLAVNLYQA                         GQDFLMLNGD                                 FSSSLLNLSQ                                         LNIGLEGVYQ    251  QENAALALQT                 FLLFMGEGKE                         AVDEQAVRKA                                 LEQTHWAGRL                                         ERIRPQIYLD    301  GAHNLPALTR                 LVEFIKEKEQ                         EGYRPQILFG                                 ALKRKDYQGM                                         LGYLTEKLPQ    351  VELKVTGFDY                 QGALDERDVT                         GYDIVSSYRE                                 FISDFEEPAD                                         AQDLLFVTGS    401  LYFISEV -COOH    __________________________________________________________________________

Deposited materials

A deposit containing a Streptococcus pneumoniae 0100993 strain has beendeposited with the National Collections of Industrial and MarineBacteria Ltd. (herein "NCIMB"), 23 St. Machar Drive, Aberdeen AB2 IRY,Scotland on 11 Apr. 1996 and assigned deposit number 40794. The depositwas described as Streptococcus peumnoiae 0100993 on deposit. On 17 Apr.1996 a Streptococcus peumnoiae 0100993 DNA library in E. coli wassimilarly depositedwith the NCIMB and assigned deposit number 40800. TheStreptococcus pneumoniae strain deposit is referred to herein as "thedeposited strain" or as "the DNA of the deposited strain."

The deposited strain contains the full length folC gene. The sequence ofthe polynucleotides contained in the deposited strain, as well as theamino acid sequence of the polypeptide encoded thereby, are controllingin the event of any conflict with any description of sequences herein.

The deposit of the deposited strain has been made under the terms of theBudapest Treaty on the International Recognition of the Deposit ofMicro-organisms for Purposes of Patent Procedure. The strain will beirrevocably and without restriction or condition released to the publicupon the issuance of a patent. The deposited strain is provided merelyas convenience to those of skill in the art and is not an admission thata deposit is required for enablement, such as that required under 35U.S.C. §112.

A license may be required to make, use or sell the deposited strain, andcompounds derived therefrom, and no such license is hereby granted.

Polypeptides

The polypeptides of the invention include the polypeptide of Table 1 SEQID NO:2! (in particular the mature polypeptide) as well as polypeptidesand fragments, particularly those which have the biological activity offolC, and also those which have at least 70% identity to a polypeptideof Table 1 SEQ ID NOS:2 and 4! or the relevant portion, preferably atleast 80% identity to a polypeptide of Table 1 SEQ ID NOS:2 and 4!, andmore preferably at least 90% similarity (more preferably at least 90%identity) to a polypeptide of Table 1 SEQ ID NOS:2 and 4! and still morepreferably at least 95% similarity (still more preferably at least 95%identity) to a polypeptide of Table 1 SEQ ID NOS:2 and 4! and alsoinclude portions of such polypeptides with such portion of thepolypeptide generally containing at least 30 amino acids and morepreferably at least 50 amino acids.

The invention also includes polypeptides of the formula set forth inTable 1 (D) SEQ ID NO:2! wherein, at the amino terminus, X is hydrogen,and at the carboxyl terminus, Y is hydrogen or a metal, R₁ and R₂ is anyamino acid residue, and n is an integer between 1 and 1000. Any stretchof amino acid residues denoted by either R group, where n is greaterthan 1, may be either a heteropolymer or a homopolymer, preferably aheteropolymer.

A fragment is a variant polypeptide having an amino acid sequence thatentirely is the same as part but not all of the amino acid sequence ofthe aforementioned polypeptides. As with folC polypeptides fragments maybe "free-standing," or comprised within a larger polypeptide of whichthey form a part or region, most preferably as a single continuousregion, a single larger polypeptide.

Preferred fragments include, for example, truncation polypeptides havinga portion of an amino acid sequence of Table 1 SEQ ID NOS:2 and 4!, orof variants thereof, such as a continuous series of residues thatincludes the amino terminus, or a continuous series of residues thatincludes the carboxyl terminus. Degradation forms of the polypeptides ofthe invention in a host cell, particularly a Streptococcus pneumoniae,are also preferred. Further preferred are fragments characterized bystructural or functional attributes such as fragments that comprisealpha-helix and alpha-helix forming regions, beta-sheet andbeta-sheet-forming regions, turn and turn-forming regions, coil andcoil-forming regions, hydrophilic regions, hydrophobic regions, alphaamphipathic regions, beta amphipathic regions, flexible regions,surface-forming regions, substrate binding region, and high antigenicindex regions.

Also preferred are biologically active fragments which are thosefragments that mediate activities of folC, including those with asimilar activity or an improved activity, or with a decreasedundesirable activity. Also included are those fragments that areantigenic or immunogenic in an animal, especially in a human.Particularly preferred are fragments comprising receptors or domains ofenzymes that confer a function essential for viability of Streptococcuspneumoniae or the ability to initiate, or maintain cause disease in anindividual, particularly a human.

Variants that are fragments of the polypeptides of the invention may beemployed for producing the corresponding full-length polypeptide bypeptide synthesis; therefore, these variants may be employed asintermediates for producing the full-length polypeptides of theinvention.

Polynucleotides

Another aspect of the invention relates to isolated polynucleotides,including the full length gene, that encode the folC polypeptide havinga deduced amino acid sequence of Table 1 SEQ ID NOS:2 and 4! andpolynucleotides closely related thereto and variants thereof.

Using the information provided herein, such as a polynucleotide sequenceset out in Table 1 SEQ ID NOS: 1 and 3!, a polynucleotide of theinvention encoding folC polypeptide may be obtained using standardcloning and screening methods, such as those for cloning and sequencingchromosomal DNA fragments from bacteria using Streptococcus pneumoniae0100993 cells as starting material, followed by obtaining a full lengthclone. For example, to obtain a polynucleotide sequence of theinvention, such as a sequence given in Table 1 SEQ ID NOS: 1 and 3!,typically a library of clones of chromosomal DNA of Streptococcuspneumoniae 0100993 in E.coli or some other suitable host is probed witha radiolabeled oligonucleotide, preferably a 17-mer or longer, derivedfrom a partial sequence. Clones carrying DNA identical to that of theprobe can then be distinguished using stringent conditions. Bysequencing the individual clones thus identified with sequencing primersdesigned from the original sequence it is then possible to extend thesequence in both directions to determine the full gene sequence.Conveniently, such sequencing is performed using denatured doublestranded DNA prepared from a plasmid clone. Suitable techniques aredescribed by Maniatis, T., Fritsch, E. F. and Sambrook et al., MOLECULARCLONING, A LABORATORY A L, 2nd Ed.; Cold Spring Harbor Laboratory Press,Cold Spring Harbor, New York (1989). (see in particular Screening ByHybridization 1.90 and Sequencing Denatured Double-Stranded DNATemplates 13.70). Illustrative of the invention, the polynucleotide setout in Table 1 SEQ ID NO:1! was discovered in a DNA library derived fromStreptococcus pneumoniae 0100993.

The DNA sequence set out in Table 1 SEQ ID NOS: 1! contains an openreading frame encoding a protein having about the number of amino acidresidues set forth in Table 1 SEQ ID NO:2! with a deduced molecularweight that can be calculated using amino acid residue molecular weightvalues well known in the art. The polynucleotide of SEQ ID NO:1, betweennucleotide number 1 through number 1254 encodes the polypeptide of SEQID NO:2. The stop codon begins at nucleotide number 1255 of SEQ ID NO:1

FolC of the invention is structurally related to other proteins of thefpgs family, as shown by the results of sequencing the DNA encoding folCof the deposited strain. The protein exhibits greatest homology to sulBprotein among known proteins. FolC of Table 1 SEQ ID NO:2! has about 39%identity over its entire length and about 63% similarity over its entirelength with the amino acid sequence of sulB polypeptide.

The invention provides a polynucleotide sequence identical over itsentire length to the coding sequence in Table 1 SEQ ID NO:1!. Alsoprovided by the invention is the coding sequence for the maturepolypeptide or a fragment thereof, by itself as well as the codingsequence for the mature polypeptide or a fragment in reading frame withother coding sequence, such as those encoding a leader or secretorysequence, a pre-, or pro- or prepro- protein sequence. Thepolynucleotide may also contain non-coding sequences, including forexample, but not limited to non-coding 5' and 3' sequences, such as thetranscribed, non-translated sequences, termination signals, ribosomebinding sites, sequences that stabilize mRNA, introns, polyadenylationsignals, and additional coding sequence which encode additional aminoacids. For example, a marker sequence that facilitates purification ofthe fused polypeptide can be encoded. In certain embodiments of theinvention, the marker sequence is a hexa-histidine peptide, as providedin the pQE vector (Qiagen, Inc.) and described in Gentz et al., Proc.Natl. Acad Sci., USA 86: 821-824 (1989), or an HA tag (Wilson et al.,Cell 37: 767 (1984). Polynucleotides of the invention also include, butare not limited to, polynucleotides comprising a structural gene and itsnaturally associated sequences that control gene expression.

A preferred embodiment of the invention is a polynucleotide ofcomprising nucleotide 1 to 1254 set forth in SEQ ID NO:1 of Table 1which encode the folC polypeptide.

The invention also includes polynucleotides of the formula set forth inTable 1 (C) SEQ ID NO:1! wherein, at the 5' end of the molecule, X ishydrogen, and at the 3' end of the molecule, Y is hydrogen or a metal,R₁ and R₂ is any nucleic acid residue, and n is an integer between 1 and1000. Any stretch of nucleic acid residues denoted by either R group,where n is greater than 1, may be either a heteropolymer or ahomopolymer, preferably a heteropolymer.

The term "polynucleotide encoding a polypeptide" as used hereinencompasses polynucleotides that include a sequence encoding apolypeptide of the invention, particularly a bacterial polypeptide andmore particularly a polypeptide of the Streptococcus pneumoniae folChaving the amino acid sequence set out in Table 1 SEQ ID NO:2!. The termalso encompasses polynucleotides that include a single continuous regionor discontinuous regions encoding the polypeptide (for example,interrupted by integrated phage or an insertion sequence or editing)together with additional regions, that also may contain coding and/ornon-coding sequences.

The invention further relates to variants of the polynucleotidesdescribed herein that encode for variants of the polypeptide having thededuced amino acid sequence of Table 1 SEQ ID NO:2!. Variants that arefragments of the polynucleotides of the invention may be used tosynthesize full-length polynucleotides of the invention.

Further particularly preferred embodiments are polynucleotides encodingfolC variants, that have the amino acid sequence of folC polypeptide ofTable 1 SEQ ID NO:2! in which several, a few, 5 to 10, 1 to 5, 1 to 3,2, 1 or no amino acid residues are substituted, deleted or added, in anycombination. Especially preferred among these are silent substitutions,additions and deletions, that do not alter the properties and activitiesof folC.

Further preferred embodiments of the invention are polynucleotides thatare at least 70% identical over their entire length to a polynucleotideencoding folC polypeptide having an amino acid sequence set out in Table1 SEQ ID NOS:2 and 4!, and polynucleotides that are complementary tosuch polynucleotides. Alternatively, most highly preferred arepolynucleotides that comprise a region that is at least 80% identicalover its entire length to a polynucleotide encoding folC polypeptide ofthe deposited strain and polynucleotides complementary thereto. In thisregard, polynucleotides at least 90% identical over their entire lengthto the same are particularly preferred, and among these particularlypreferred polynucleotides, those with at least 95% are especiallypreferred. Furthermore, those with at least 97% are highly preferredamong those with at least 95%, and among these those with at least 98%and at least 99% are particularly highly preferred, with at least 99%being the more preferred.

Preferred embodiments are polynucleotides that encode polypeptides thatretain substantially the same biological function or activity as themature polypeptide encoded by the DNA of Table 1 SEQ ID NO:1!.

The invention further relates to polynucleotides that hybridize to theherein above-described sequences. In this regard, the inventionespecially relates to polynucleotides that hybridize under stringentconditions to the herein above-described polynucleotides. As hereinused, the terms "stringent conditions" and "stringent hybridizationconditions" mean hybridization will occur only if there is at least 95%and preferably at least 97% identity between the sequences. An exampleof stringent hybridization conditions is overnight incubation at 42° Cin a solution comprising: 50% formamide, 5×SSC (150 mM NaCl, 15 mMtrisodium citrate), 50 mM sodium phosphate (pH7.6), 5×Denhardt'ssolution, 10% dextran sulfate, and 20 micrograms/ml denatured, shearedsalmon sperm DNA, followed by washing the hybridization support in0.1×SSC at about 65° C. Hybridization and wash conditions are well knownand exemplified in Sambrook, et al., Molecular Cloning: A LaboratoryManual, Second Edition, Cold Spring Harbor, N.Y., (1989), particularlyChapter 11 therein.

The invention also provides a polynucleotide consisting essentially of apolynucleotide sequence obtainable by screening an appropriate librarycontaining the complete gene for a polynucleotide sequence set forth inSEQ ID NO:1 or SEQ ID NO:3 under stringent hybridization conditions witha probe having the sequence of said polynucleotide sequence set forth inSEQ ID NO:1 or a fragment thereof; and isolating said DNA sequence.Fragments useful for obtaining such a polynucleotide include, forexample, probes and primers described elsewhere herein.

As discussed additionally herein regarding polynucleotide assays of theinvention, for instance, polynucleotides of the invention as discussedabove, may be used as a hybridization probe for RNA, cDNA and genomicDNA to isolate full-length cDNAs and genomic clones encoding folC and toisolate cDNA and genomic clones of other genes that have a high sequencesimilarity to the folC gene. Such probes generally will comprise atleast 15 bases. Preferably, such probes will have at least 30 bases andmay have at least 50 bases. Particularly preferred probes will have atleast 30 bases and will have 50 bases or less.

For example, the coding region of the folC gene may be isolated byscreening using the DNA sequence provided in SEQ ID NO:1 to synthesizean oligonucleotide probe. A labeled oligonucleotide having a sequencecomplementary to that of a gene of the invention is then used to screena library of cDNA, genomic DNA or mRNA to determine which members of thelibrary the probe hybridizes to.

The polynucleotides and polypeptides of the invention may be employed,for example, as research reagents and materials for discovery oftreatments of and diagnostics for disease, particularly human disease,as further discussed herein relating to polynucleotide assays.

Polynucleotides of the invention that are oligonucleotides derived fromthe sequences of SEQ ID NOS:1 and/or 2 may be used in the processesherein as described, but preferably for PCR, to determine whether or notthe polynucleotides identified herein in whole or in part aretranscribed in bacteria in infected tissue. It is recognized that suchsequences will also have utility in diagnosis of the stage of infectionand type of infection the pathogen has attained.

The invention also provides polynucleotides that may encode apolypeptide that is the mature protein plus additional amino orcarboxyl-terminal amino acids, or amino acids interior to the maturepolypeptide (when the mature form has more than one polypeptide chain,for instance). Such sequences may play a role in processing of a proteinfrom precursor to a mature form, may allow protein transport, maylengthen or shorten protein half-life or may facilitate manipulation ofa protein for assay or production, among other things. As generally isthe case in vivo, the additional amino acids may be processed away fromthe mature protein by cellular enzymes.

A precursor protein, having the mature form of the polypeptide fused toone or more prosequences may be an inactive form of the polypeptide.When prosequences are removed such inactive precursors generally areactivated. Some or all of the prosequences may be removed beforeactivation. Generally, such precursors are called proproteins.

In sum, a polynucleotide of the invention may encode a mature protein, amature protein plus a leader sequence (which may be referred to as apreprotein), a precursor of a mature protein having one or moreprosequences that are not the leader sequences of a preprotein, or apreproprotein, which is a precursor to a proprotein, having a leadersequence and one or more prosequences, which generally are removedduring processing steps that produce active and mature forms of thepolypeptide.

Vectors, host cells, expression

The invention also relates to vectors that comprise a polynucleotide orpolynucleotides of the invention, host cells that are geneticallyengineered with vectors of the invention and the production ofpolypeptides of the invention by recombinant techniques. Cell-freetranslation systems can also be employed to produce such proteins usingRNAs derived from the DNA constructs of the invention.

For recombinant production, host cells can be genetically engineered toincorporate expression systems or portions thereof or polynucleotides ofthe invention. Introduction of a polynucleotide into the host cell canbe effected by methods described in many standard laboratory manuals,such as Davis et al., BASIC METHODS IN MOLECULAR BIOLOGY, (1986) andSambrook et al., MOLECULAR CLONING: A LABORATORY MANUAL, 2nd Ed., ColdSpring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1989), suchas, calcium phosphate transfection, DEAE-dextran mediated transfection,transvection, microinjection, cationic lipid-mediated transfection,electroporation, transduction, scrape loading, ballistic introductionand infection.

Representative examples of appropriate hosts include bacterial cells,such as streptococci, staphylococci, enterococci E. coli, streptomycesand Bacillus subtilis cells; fungal cells, such as yeast cells andAspergillus cells; insect cells such as Drosophila S2 and Spodoptera Sf9cells; animal cells such as CHO, COS, HeLa, C127, 3T3, BHK, 293 andBowes melanoma cells; and plant cells.

A great variety of expression systems can be used to produce thepolypeptides of the invention. Such vectors include, among others,chromosomal, episomal and virus-derived vectors, e.g., vectors derivedfrom bacterial plasmids, from bacteriophage, from transposons, fromyeast episomes, from insertion elements, from yeast chromosomalelements, from viruses such as baculoviruses, papova viruses, such asSV40, vaccinia viruses, adenoviruses, fowl pox viruses, pseudorabiesviruses and retroviruses, and vectors derived from combinations thereof,such as those derived from plasmid and bacteriophage genetic elements,such as cosmids and phagemids. The expression system constructs maycontain control regions that regulate as well as engender expression.Generally, any system or vector suitable to maintain, propagate orexpress polynucleotides and/or to express a polypeptide in a host may beused for expression in this regard. The appropriate DNA sequence may beinserted into the expression system by any of a variety of well-knownand routine techniques, such as, for example, those set forth inSambrook et al., MOLECULAR CLONING, A LABORATORY MANUAL, (supra).

For secretion of the translated protein into the lumen of theendoplasmic reticulum, into the periplasmic space or into theextracellular environment, appropriate secretion signals may beincorporated into the expressed polypeptide. These signals may beendogenous to the polypeptide or they may be heterologous signals.

Polypeptides of the invention can be recovered and purified fromrecombinant cell cultures by well-known methods including ammoniumsulfate or ethanol precipitation, acid extraction, anion or cationexchange chromatography, phosphocellulose chromatography, hydrophobicinteraction chromatography, affinity chromatography, hydroxylapatitechromatography, and lectin chromatography. Most preferably, highperformance liquid chromatography is employed for purification. Wellknown techniques for refolding protein may be employed to regenerateactive conformation when the polypeptide is denatured during isolationand or purification.

Diagnostic Assays

This invention is also related to the use of the folC polynucleotides ofthe invention for use as diagnostic reagents. Detection of folC in aeukaryote, particularly a mammal, and especially a human, will provide adiagnostic method for diagnosis of a disease. Eukaryotes (herein also"individual(s)"), particularly mammals, and especially humans,particularly those infected or suspected to be infected with an organismcomprising the folC gene may be detected at the nucleic acid level by avariety of techniques.

Nucleic acids for diagnosis may be obtained from an infectedindividual's cells and tissues, such as bone, blood, muscle, cartilage,and skin. Genomic DNA may be used directly for detection or may beamplified enzymatically by using PCR or other amplification techniqueprior to analysis. RNA or cDNA may also be used in the same ways. Usingamplification, characterization of the species and strain of prokaryotepresent in an individual, may be made by an analysis of the genotype ofthe prokaryote gene. Deletions and insertions can be detected by achange in size of the amplified product in comparison to the genotype ofa reference sequence. Point mutations can be identified by hybridizingamplified DNA to labeled folC polynucleotide sequences. Perfectlymatched sequences can be distinguished from mismatched duplexes by RNasedigestion or by differences in melting temperatures. DNA sequencedifferences may also be detected by alterations in the electrophoreticmobility of the DNA fragments in gels, with or without denaturingagents, or by direct DNA sequencing. See, e.g. Myers et al., Science,230: 1242 (1985). Sequence changes at specific locations also may berevealed by nuclease protection assays, such as RNase and S1 protectionor a chemical cleavage method. See, e.g. Cotton et al., Proc. Natl.Acad. Sci., USA, 85: 4397-4401 (1985).

Cells carrying mutations or polymorphisms in the gene of the inventionmay also be detected at the DNA level by a variety of techniques, toallow for serotyping, for example. For example, RT-PCR can be used todetect mutations. It is particularly preferred to used RT-PCR inconjunction with automated detection systems, such as, for example,GeneScan. RNA or cDNA may also be used for the same purpose, PCR orRT-PCR. As an example, PCR primers complementary to a nucleic acidencoding folC can be used to identify and analyze mutations.

The invention further provides these primers with 1, 2, 3 or 4nucleotides removed from the 5' and/or the 3' end. These primers may beused for, among other things, amplifying folC DNA isolated from a samplederived from an individual. The primers may be used to amplify the geneisolated from an infected individual such that the gene may then besubject to various techniques for elucidation of the DNA sequence. Inthis way, mutations in the DNA sequence may be detected and used todiagnose infection and to serotype and/or classify the infectious agent.

The invention further provides a process for diagnosing, disease,preferably bacterial infections, more preferably infections byStreptococcus pneumoniae, and most preferably otitis media,conjunctivitis, pneumonia, bacteremia, meningitis, sinusitis, pleuralempyema and endocarditis, and most particularly meningitis, such as forexample infection of cerebrospinal fluid, comprising determining from asample derived from an individual a increased level of expression ofpolynucleotide having the sequence of Table 1 SEQ ID NO:1!. Increased ordecreased expression of folC polynucleotide can be measured using any onof the methods well known in the art for the quantation ofpolynucleotides, such as, for example, amplification, PCR, RT-PCR, RNaseprotection, Northern blotting and other hybridization methods.

In addition, a diagnostic assay in accordance with the invention fordetecting over-expression of folC protein compared to normal controltissue samples may be used to detect the presence of an infection, forexample. Assay techniques that can be used to determine levels of a folCprotein, in a sample derived from a host are well-known to those ofskill in the art. Such assay methods include radioimmunoassays,competitive-binding assays, Western Blot analysis and ELISA assays.

Antibodies

The polypeptides of the invention or variants thereof, or cellsexpressing them can be used as an immunogen to produce antibodiesimmunospecific for such polypeptides. "Antibodies" as used hereinincludes monoclonal and polyclonal antibodies, chimeric, single chain,simianized antibodies and humanized antibodies, as well as Fabfragments, including the products of an Fab immunolglobulin expressionlibrary.

Antibodies generated against the polypeptides of the invention can beobtained by administering the polypeptides or epitope-bearing fragments,analogues or cells to an animal, preferably a nonhuman, using routineprotocols. For preparation of monoclonal antibodies, any technique knownin the art that provides antibodies produced by continuous cell linecultures can be used. Examples include various techniques, such as thosein Kohler, G. and Milstein, C., Nature 256: 495-497 (1975); Kozbor etal., Immunology Today 4: 72 (1983); Cole et al., pg. 77-96 in MONOCLONALANTIBODIES AND CANCER THERAPY, Alan R. Liss, Inc. (1985).

Techniques for the production of single chain antibodies (U.S. Pat. No.4,946,778) can be adapted to produce single chain antibodies topolypeptides of this invention. Also, transgenic mice, or otherorganisms such as other mammals, may be used to express humanizedantibodies.

Alternatively phage display technology may be utilized to selectantibody genes with binding activities towards the polypeptide eitherfrom repertoires of PCR amplified v-genes of lymphocytes from humansscreened for possessing anti-folC or from naive libraries (McCafferty,J. et al., (1990), Nature 348, 552-554; Marks, J. et al., (1992)Biotechnology 10, 779-783). The affinity of these antibodies can also beimproved by chain shuffling (Clackson, T. et al., (1991) Nature 352,624-628).

If two antigen binding domains are present each domain may be directedagainst a different epitope--termed `bispecific` antibodies.

The above-described antibodies may be employed to isolate or to identifyclones expressing the polypeptides to purify the polypeptides byaffinity chromatography.

Thus, among others, antibodies against folC- polypeptide may be employedto treat infections, particularly bacterial infections and especiallyotitis media, conjunctivitis, pneumonia, bacteremia, meningitis,sinusitis, pleural empyema and endocarditis, and most particularlymeningitis, such as for example infection of cerebrospinal fluid.

Polypeptide variants include antigenically, epitopically orimmunologically equivalent variants that form a particular aspect ofthis invention. The term "antigenically equivalent derivative" as usedherein encompasses a polypeptide or its equivalent which will bespecifically recognized by certain antibodies which, when raised to theprotein or polypeptide according to the invention, interfere with theimmediate physical interaction between pathogen and mammalian host. Theterm "immunologically equivalent derivative" as used herein encompassesa peptide or its equivalent which when used in a suitable formulation toraise antibodies in a vertebrate, the antibodies act to interfere withthe immediate physical interaction between pathogen and mammalian host.

The polypeptide, such as an antigenically or immunologically equivalentderivative or a fusion protein thereof is used as an antigen to immunizea mouse or other animal such as a rat or chicken. The fusion protein mayprovide stability to the polypeptide. The antigen may be associated, forexample by conjugation, with an immunogenic carrier protein for examplebovine serum albumin (BSA) or keyhole limpet haemocyanin (KLH).Alternatively a multiple antigenic peptide comprising multiple copies ofthe protein or polypeptide, or an antigenically or immunologicallyequivalent polypeptide thereof may be sufficiently antigenic to improveimmunogenicity so as to obviate the use of a carrier.

Preferably, the antibody or variant thereof is modified to make it lessimmunogenic in the individual. For example, if the individual is humanthe antibody may most preferably be "humanized"; where thecomplimentarity determining region(s) of the hybridoma-derived antibodyhas been transplanted into a human monoclonal antibody, for example asdescribed in Jones, P. et al. (1986), Nature 321, 522-525 or Tempest etal., (1991) Biotechnology 9, 266-273.

The use of a polynucleotide of the invention in genetic immunizationwill preferably employ a suitable delivery method such as directinjection of plasmid DNA into muscles (Wolff et al., Hum Mol Genet 1992,1:363, Manthorpe et al., Hum. Gene Ther. 1963:4, 419), delivery of DNAcomplexed with specific protein carriers (Wu et al., J Biol Chem. 1989:264,16985), coprecipitation of DNA with calcium phosphate (13envenisty &Reshef, PNAS USA, 1986:83,9551), encapsulation of DNA in various formsof liposomes (Kaneda et al., Science 1989:243,375), particle bombardment(Tang et al., Nature 1992, 356:152, Eisenbraun et al., DNA Cell Biol1993, 12:791) and in vivo infection using cloned retroviral vectors(Seeger et al., PNAS USA 1984:81,5849).

Antagonists and agonists--assays and molecules

Polypeptides of the invention may also be used to assess the binding ofsmall molecule substrates and ligands in, for example, cells, cell-freepreparations, chemical libraries, and natural product mixtures. Thesesubstrates and ligands may be natural substrates and ligands or may bestructural or functional mimetics. See, e.g., Coligan et al., CurrentProtocols in Immunology 1(2): Chapter 5 (1991).

The invention also provides a method of screening compounds to identifythose which enhance (agonist) or block (antagonist) the action of folCpolypeptides or polynucleotides, particularly those compounds that arebacteriostatic and/or bacteriocidal. The method of screening may involvehigh-throughput techniques. For example, to screen for agonists orantagoists, a synthetic reaction mix, a cellular compartment, such as amembrane, cell envelope or cell wall, or a preparation of any thereof,comprising folC polypeptide and a labeled substrate or ligand of suchpolypeptide is incubated in the absence or the presence of a candidatemolecule that may be a folC agonist or antagonist. The ability of thecandidate molecule to agonize or antagonize the folC polypeptide isreflected in decreased binding of the labeled ligand or decreasedproduction of product from such substrate. Molecules that bindgratuitously, i.e., without inducing the effects of folC polypeptide aremost likely to be good antagonists. Molecules that bind well andincrease the rate of product production from substrate are agonists.Detection of the rate or level of production of product from substratemay be enhanced by using a reporter system. Reporter systems that may beuseful in this regard include but are not limited to colorimetriclabeled substrate converted into product, a reporter gene that isresponsive to changes in folC polynucleotide or polypeptide activity,and binding assays known in the art.

Another example of an assay for folC antagonists is a competitive assaythat combines folC and a potential antagonist with foIC-bindingmolecules, recombinant folC binding molecules, natural substrates orligands, or substrate or ligand mimetics, under appropriate conditionsfor a competitive inhibition assay. FolC can be labeled, such as byradioactivity or a calorimetric compound, such that the number of folCmolecules bound to a binding molecule or converted to product can bedetermined accurately to assess the effectiveness of the potentialantagonist.

Potential antagonists include small organic molecules, peptides,polypeptides and antibodies that bind to a polynucleotide or polypeptideof the invention and thereby inhibit or extinguish its activity.Potential antagonists also may be small organic molecules, a peptide, apolypeptide such as a closely related protein or antibody that binds thesame sites on a binding molecule, such as a binding molecule, withoutinducing folC-induced activities, thereby preventing the action of folCby excluding folC from binding.

Potential antagonists include a small molecule that binds to andoccupies the binding site of the polypeptide thereby preventing bindingto cellular binding molecules, such that normal biological activity isprevented. Examples of small molecules include but are not limited tosmall organic molecules, peptides or peptide-like molecules. Otherpotential antagonists include antisense molecules (see Okano, JNeurochem. 56: 560 (1991); OLIGODEOXYNUCLEOTIDES AS ANTISENSE INHIBITORSOF GENE EXPRESSION, CRC Press, Boca Raton, Fla. (1988), for adescription of these molecules). Preferred potential antagonists includecompounds related to and variants of folC.

Each of the DNA sequences provided herein may be used in the discoveryand development of antibacterial compounds. The encoded protein, uponexpression, can be used as a target for the screening of antibacterialdrugs. Additionally, the DNA sequences encoding the amino terminalregions of the encoded protein or Shine-Delgarno or other translationfacilitating sequences of the respective mRNA can be used to constructantisense sequences to control the expression of the coding sequence ofinterest.

The invention also provides the use of the polypeptide, polynucleotideor inhibitor of the invention to interfere with the initial physicalinteraction between a pathogen and mammalian host responsible forsequelae of infection. In particular the molecules of the invention maybe used: in the prevention of adhesion of bacteria, in particular grampositive bacteria, to mammalian extracellular matrix proteins onin-dwelling devices or to extracellular matrix proteins in wounds; toblock folC protein-mediated mammalian cell invasion by, for example,initiating phosphorylation of mammalian tyrosine kinases (Rosenshine etal., Infect. Immun. 60:2211 (1992); to block bacterial adhesion betweenmammalian extracellular matrix proteins and bacterial folC proteins thatmediate tissue damage and; to block the normal progression ofpathogenesis in infections initiated other than by the implantation ofin-dwelling devices or by other surgical techniques.

The antagonists and agonists of the invention may be employed, forinstance, to inhibit and treat otitis media, conjunctivitis, pneumonia,bacteremia, meningitis, sinusitis, pleural empyema and endocarditis, andmost particularly meningitis, such as for example infection ofcerebrospinal fluid.

Helicobacter pylori (herein H. pylori) bacteria infect the stomachs ofover one-third of the world's population causing stomach cancer, ulcers,and gastritis (International Agency for Research on Cancer (1994)Schistosomes, Liver Flukes and Helicobacter Pylori (International Agencyfor Research on Cancer, Lyon, France;http://www.uicc.ch/ecp/ecp2904.htm). Moreover, the international Agencyfor Research on Cancer recently recognized a cause-and-effectrelationship between H. pylori and gastric adenocarcinoma, classifyingthe bacterium as a Group I (definite) carcinogen. Preferredantimicrobial compounds of the invention (agonists and antagonists offolC) found using screens provided by the invention, particularlybroad-spectrum antibiotics, should be useful in the treatment of H.pylori infection. Such treatment should decrease the advent of H.pylori-induced cancers, such as gastrointestinal carcinoma. Suchtreatment should also cure gastric ulcers and gastritis.

Vaccines

Another aspect of the invention relates to a method for inducing animmunological response in an individual, particularly a mammal whichcomprises inoculating the individual with folC, or a fragment or variantthereof, adequate to produce antibody and/ or T cell immune response toprotect said individual from infection, particularly bacterial infectionand most particularly Streptococcus pneumoniae infection. Also providedare methods whereby such immunological response slows bacterialreplication. Yet another aspect of the invention relates to a method ofinducing immunological response in an individual which comprisesdelivering to such individual a nucleic acid vector to direct expressionof folC, or a fragment or a variant thereof, for expressing folC, or afragment or a variant thereof in vivo in order to induce animmunological response, such as, to produce antibody and/ or T cellimmune response, including, for example, cytokine-producing T cells orcytotoxic T cells, to protect said individual from disease, whether thatdisease is already established within the individual or not. One way ofadministering the gene is by accelerating it into the desired cells as acoating on particles or otherwise. Such nucleic acid vector may compriseDNA, RNA, a modified nucleic acid, or a DNA/RNA hybrid.

A further aspect of the invention relates to an immunologicalcomposition which, when introduced into an individual capable or havinginduced within it an immunological response, induces an immunologicalresponse in such individual to a folC or protein coded therefrom,wherein the composition comprises a recombinant folC or protein codedtherefrom comprising DNA which codes for and expresses an antigen ofsaid folC or protein coded therefrom. The immunological response may beused therapeutically or prophylactically and may take the form ofantibody immunity or cellular immunity such as that arising from CTL orCD4+T cells.

A folC polypeptide or a fragment thereof may be fused with co-proteinwhich may not by itself produce antibodies, but is capable ofstabilizing the first protein and producing a fused protein which willhave immunogenic and protective properties. Thus fused recombinantprotein, preferably further comprises an antigenic co-protein, such aslipoprotein D from Hemophilus influenzae, Glutathione-S-transferase(GST) or beta-galactosidase, relatively large co-proteins whichsolubilize the protein and facilitate production and purificationthereof. Moreover, the co-protein may act as an adjuvant in the sense ofproviding a generalized stimulation of the immune system. The co-proteinmay be attached to either the amino or carboxy terminus of the firstprotein.

Provided by this invention are compositions, particularly vaccinecompositions, and methods comprising the polypeptides or polynucleotidesof the invention and immunostimulatory DNA sequences, such as thosedescribed in Sato, Y. et al. Science 273: 352 (1996).

Also, provided by this invention are methods using the describedpolynucleotide or particular fragments thereof which have been shown toencode non-variable regions of bacterial cell surface proteins in DNAconstructs used in such genetic immunization experiments in animalmodels of infection with Streptococcus pneumoniae will be particularlyuseful for identifying protein epitopes able to provoke a prophylacticor therapeutic immune response. It is believed that this approach willallow for the subsequent preparation of monoclonal antibodies ofparticular value from the requisite organ of the animal successfullyresisting or clearing infection for the development of prophylacticagents or therapeutic treatments of bacterial infection, particularlyStreptococcus pneumoniae infection, in mammals, particularly humans.

The polypeptide may be used as an antigen for vaccination of a host toproduce specific antibodies which protect against invasion of bacteria,for example by blocking adherence of bacteria to damaged tissue.Examples of tissue damage include wounds in skin or connective tissuecaused, e.g., by mechanical, chemical or thermal damage or byimplantation of indwelling devices, or wounds in the mucous membranes,such as the mouth, mammary glands, urethra or vagina.

The invention also includes a vaccine formulation which comprises animmunogenic recombinant protein of the invention together with asuitable carrier. Since the protein may be broken down in the stomach,it is preferably administered parenterally, including, for example,administration that is subcutaneous, intramuscular, intravenous, orintradermal. Formulations suitable for parenteral administration includeaqueous and non-aqueous sterile injection solutions which may containanti-oxidants, buffers, bacteriostats and solutes which render theformulation insotonic with the bodily fluid, preferably the blood, ofthe individual; and aqueous and non-aqueous sterile suspensions whichmay include suspending agents or thickening agents. The formulations maybe presented in unit-dose or multi-dose containers, for example, sealedampules and vials and may be stored in a freeze-dried conditionrequiring only the addition of the sterile liquid carrier immediatelyprior to use. The vaccine formulation may also include adjuvant systemsfor enhancing the immunogenicity of the formulation, such as oil-inwater systems and other systems known in the art. The dosage will dependon the specific activity of the vaccine and can be readily determined byroutine experimentation.

While the invention has been described with reference to certain folCprotein, it is to be understood that this covers fragments of thenaturally occurring protein and similar proteins with additions,deletions or substitutions which do not substantially affect theimmunogenic properties of the recombinant protein.

Compositions, kits and administration

The invention also relates to compositions comprising the polynucleotideor the polypeptides discussed above or their agonists or antagonists.The polypeptides of the invention may be employed in combination with anon-sterile or sterile carrier or carriers for use with cells, tissuesor organisms, such as a pharmaceutical carrier suitable foradministration to a subject. Such compositions comprise, for instance, amedia additive or a therapeutically effective amount of a polypeptide ofthe invention and a pharmaceutically acceptable carrier or excipient.Such carriers may include, but are not limited to, saline, bufferedsaline, dextrose, water, glycerol, ethanol and combinations thereof. Theformulation should suit the mode of administration. The inventionfurther relates to diagnostic and pharmaceutical packs and kitscomprising one or more containers filled with one or more of theingredients of the aforementioned compositions of the invention.

Polypeptides and other compounds of the invention may be employed aloneor in conjunction with other compounds, such as therapeutic compounds.

The pharmaceutical compositions may be administered in any effective,convenient manner including, for instance, administration by topical,oral, anal, vaginal, intravenous, intraperitoneal, intramuscular,subcutaneous, intranasal or intradermal routes among others.

In therapy or as a prophylactic, the active agent may be administered toan individual as an injectable composition, for example as a sterileaqueous dispersion, preferably isotonic.

Alternatively the composition may be formulated for topical applicationfor example in the form of ointments, creams, lotions, eye ointments,eye drops, ear drops, mouthwash, impregnated dressings and sutures andaerosols, and may contain appropriate conventional additives, including,for example, preservatives, solvents to assist drug penetration, andemollients in ointments and creams. Such topical formulations may alsocontain compatible conventional carriers, for example cream or ointmentbases, and ethanol or oleyl alcohol for lotions. Such carriers mayconstitute from about 1% to about 98% by weight of the formulation; moreusually they will constitute up to about 80% by weight of theformulation.

For administration to mammals, and particularly humans, it is expectedthat the daily dosage level of the active agent will be from 0.01 mg/kgto 10 mg/kg, typically around 1 mg/kg. The physician in any event willdetermine the actual dosage which will be most suitable for anindividual and will vary with the age, weight and response of theparticular individual. The above dosages are exemplary of the averagecase. There can, of course, be individual instances where higher orlower dosage ranges are merited, and such are within the scope of thisinvention.

In-dwelling devices include surgical implants, prosthetic devices andcatheters, i.e., devices that are introduced to the body of anindividual and remain in position for an extended time. Such devicesinclude, for example, artificial joints, heart valves, pacemakers,vascular grafts, vascular catheters, cerebrospinal fluid shunts, urinarycatheters, continuous ambulatory peritoneal dialysis (CAPD) catheters.

The composition of the invention may be administered by injection toachieve a systemic effect against relevant bacteria shortly beforeinsertion of an in-dwelling device. Treatment may be continued aftersurgery during the in-body time of the device. In addition, thecomposition could also be used to broaden perioperative cover for anysurgical technique to prevent bacterial wound infections, especiallyStreptococcus pneumoniae wound infections.

Many orthopaedic surgeons consider that humans with prosthetic jointsshould be considered for antibiotic prophylaxis before dental treatmentthat could produce a bacteremia. Late deep infection is a seriouscomplication sometimes leading to loss of the prosthetic joint and isaccompanied by significant morbidity and mortality. It may therefore bepossible to extend the use of the active agent as a replacement forprophylactic antibiotics in this situation.

In addition to the therapy described above, the compositions of thisinvention may be used generally as a wound treatment agent to preventadhesion of bacteria to matrix proteins exposed in wound tissue and forprophylactic use in dental treatment as an alternative to, or inconjunction with, antibiotic prophylaxis.

Alternatively, the composition of the invention may be used to bathe anindwelling device immediately before insertion. The active agent willpreferably be present at a concentration of 1 μg/ml to 10 mg/ml forbathing of wounds or indwelling devices.

A vaccine composition is conveniently in injectable form. Conventionaladjuvants may be employed to enhance the immune response. A suitableunit dose for vaccination is 0.5-5 microgram/kg of antigen, and suchdose is preferably administered 1-3 times and with an interval of 1-3weeks. With the indicated dose range, no adverse toxicological effectswill be observed with the compounds of the invention which wouldpreclude their administration to suitable individuals.

Each reference disclosed herein is incorporated by reference herein inits entirety. Any patent application to which this application claimspriority is also incorporated by reference herein in its entirety.

EXAMPLES

The examples below are carried out using standard techniques, which arewell known and routine to those of skill in the art, except whereotherwise described in detail. The examples are illustrative, but do notlimit the invention.

Example 1 Strain selection, Library Production and Sequencing

The polynucleotide having the DNA sequence given in SEQ ID NO:1 wasobtained from a library of clones of chromosomal DNA of Streptococcuspneumoniae in E. coli. The sequencing data from two or more clonescontaining overlapping Streptococcus pneumoniae DNAs was used toconstruct the contiguous DNA sequence in SEQ ID NO:1. Libraries may beprepared by routine methods, for example:

Methods 1 and 2 below.

Total cellular DNA is isolated from Streptococcus pneumoniae 0100993according to standard procedures and size-fractionated by either of twomethods.

Method 1

Total cellular DNA is mechanically sheared by passage through a needlein order to size-fractionate according to standard procedures. DNAfragments of up to 11 kbp in size are rendered blunt by treatment withexonuclease and DNA polymerase, and EcoRI linkers added. Fragments areligated into the vector Lambda ZapII that has been cut with EcoRI, thelibrary packaged by standard procedures and E.coli infected with thepackaged library. The library is amplified by standard procedures.

Method 2

Total cellular DNA is partially hydrolyzed with a one or a combinationof restriction enzymes appropriate to generate a series of fragments forcloning into library vectors (e.g., RsaI, PalI, AluI, Bshl235I), andsuch fragments are size-fractionated according to standard procedures.EcoRI linkers are ligated to the DNA and the fragments then ligated intothe vector Lambda ZapII that have been cut with EcoRI, the librarypackaged by standard procedures, and E. coli infected with the packagedlibrary. The library is amplified by standard procedures.

    __________________________________________________________________________    SEQUENCE LISTING    (1) GENERAL INFORMATION:    (iii) NUMBER OF SEQUENCES: 4    (2) INFORMATION FOR SEQ ID NO:1:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 1254 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: double    (D) TOPOLOGY: linear    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:    ATGTGTATGTTTGAAGTAGAAGAATGCTTTCATAGTCGGATTGGTTTGAATTTTCGATCA60    GGTTTGGCCGGAATACAGCAAGCGGTGGATTTGTTAGGAAATCCTGAGCAGTCTTACCCT120    ATTATCCACGTAACAGGGACTAATGGGAAAGGATCTACCATTGCTTTTATGAGGGAATTA180    TTTATGGGGCATGGCAAAAAAGTTGCGACCTTTACCTCCCCTCATATCGTATCCATCAAT240    GACCGAATTTGCATTAATGGGCAACCTATAGCAGACGCAGACTTTATCCGTTTGGCTGAT300    CAGGTCAAGGAGATGGAGAAAACGCTTCTGCAAACTCCTGACCAGTTGTCCTTTTTTGAA360    TTGCTGACCTTGGTTGCTTTTCTTTATTTTAGGGAGCAGGAGGTGGATTTGGTTTTATTA420    GAAGTGGGAATTGGTGGCTTACTTGACACGACCAATGTGGTAACTGGAGAGCTTGCTGTC480    ATCACCTCCATTGGGCTTGACCATCAAGAAACCTTGGGTGATAGTCTAGAAGCAATTGCA540    GAGCAGAAAGCTGGTATTTTCAAGGCTGGTAAGAAGGCAGTGATTGCGAAATTGCCTCCA600    GAAGCTAGGCTTGTCTGTCAGAAAAAAGCCGAATCTTTAGCTGTTAACCTTTATCAGGCA660    GGTCAAGATTTTTTAATGCTGAATGGTGATTTTTCAAGCTCTTTACTAAATCTTTCACAG720    CTGAACATAGGCTTAGAAGGAGTCTATCAGCAGGAGAATGCAGCCTTGGCGTTGCAAACT780    TTTCTTCTTTTTATGGGAGAAGGAAAGGAAGCTGTTGATGAACAGGCTGTAAGAAAGGCC840    TTGGAACAGACCCATTGGGCTGGTCGCTTGGAGCGTATTCGCCCACAGATTTACTTGGAT900    GGTGCTCATAACCTCCCTGCCTTGACTCGCTTGGTTGAGTTTATCAAAGAAAAAGAGCAG960    GAAGGTTATCGTCCTCAAATCCTTTTTGGAGCCTTGAAACGGAAGGATTATCAAGGGATG1020    TTAGGTTACCTGACTGAAAAATTGCCTCAGGTGGAACTCAAGGTGACCGGCTTTGACTAT1080    CAGGGGGCTTTGGACGAAAGGGATGTGACAGGTTACGATATAGTTTCTTCTTACCGAGAA1140    TTTATCAGCGATTTTGAAGAAAGAGCAGATGCTCAGGATTTGCTGTTCGTTACAGGGTCT1200    CTCTATTTTATCTCAGAAGTACGGGGCTACCTGCTGGACCGTGAGCAGATAAAT1254    (2) INFORMATION FOR SEQ ID NO:2:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 418 amino acids    (B) TYPE: amino acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:    MetCysMetPheGluValGluGluCysPheHisSerArgIleGlyLeu    151015    AsnPheArgSerGlyLeuAlaGlyIleGlnGlnAlaValAspLeuLeu    202530    GlyAsnProGluGlnSerTyrProIleIleHisValThrGlyThrAsn    354045    GlyLysGlySerThrIleAlaPheMetArgGluLeuPheMetGlyHis    505560    GlyLysLysValAlaThrPheThrSerProHisIleValSerIleAsn    65707580    AspArgIleCysIleAsnGlyGlnProIleAlaAspAlaAspPheIle    859095    ArgLeuAlaAspGlnValLysGluMetGluLysThrLeuLeuGlnThr    100105110    ProAspGlnLeuSerPhePheGluLeuLeuThrLeuValAlaPheLeu    115120125    TyrPheArgGluGlnGluValAspLeuValLeuLeuGluValGlyIle    130135140    GlyGlyLeuLeuAspThrThrAsnValValThrGlyGluLeuAlaVal    145150155160    IleThrSerIleGlyLeuAspHisGlnGluThrLeuGlyAspSerLeu    165170175    GluAlaIleAlaGluGlnLysAlaGlyIlePheLysAlaGlyLysLys    180185190    AlaValIleAlaLysLeuProProGluAlaArgLeuValCysGlnLys    195200205    LysAlaGluSerLeuAlaValAsnLeuTyrGlnAlaGlyGlnAspPhe    210215220    LeuMetLeuAsnGlyAspPheSerSerSerLeuLeuAsnLeuSerGln    225230235240    LeuAsnIleGlyLeuGluGlyValTyrGlnGlnGluAsnAlaAlaLeu    245250255    AlaLeuGlnThrPheLeuLeuPheMetGlyGluGlyLysGluAlaVal    260265270    AspGluGlnAlaValArgLysAlaLeuGluGlnThrHisTrpAlaGly    275280285    ArgLeuGluArgIleArgProGlnIleTyrLeuAspGlyAlaHisAsn    290295300    LeuProAlaLeuThrArgLeuValGluPheIleLysGluLysGluGln    305310315320    GluGlyTyrArgProGlnIleLeuPheGlyAlaLeuLysArgLysAsp    325330335    TyrGlnGlyMetLeuGlyTyrLeuThrGluLysLeuProGlnValGlu    340345350    LeuLysValThrGlyPheAspTyrGlnGlyAlaLeuAspGluArgAsp    355360365    ValThrGlyTyrAspIleValSerSerTyrArgGluPheIleSerAsp    370375380    PheGluGluArgAlaAspAlaGlnAspLeuLeuPheValThrGlySer    385390395400    LeuTyrPheIleSerGluValArgGlyTyrLeuLeuAspArgGluGln    405410415    IleAsn    (2) INFORMATION FOR SEQ ID NO:3:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 1221 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: double    (D) TOPOLOGY: linear    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:    ATGTGTATGTTTGAAGTAGAAGAATGCTTTCATAGTCGGATTGGTTTGAATTTTCGATCA60    GGTTTGGCCGGAATACAGCAAGCGGTGGATTTGTTAGGAAATCCTGAGCAGTCTTACCCT120    ATTATCCACGTAACAGGGACTAATGGGAAAGGATCTACCATTGCTTTTATGAGGGAATTA180    TTTATGGGGCATGGCAAAAAAGTTGCGACCTTTACCTCCCCTCATATCGTATCCATCAAT240    GACCGAATTTGCATTAATGGGCAACCTATAGCAGACGCAGACTTTATCCGTTTGGCTGAT300    CAGGTCAAGGAGATGGAGAAAACGCTTCTGCAAACTCCTGACCAGTTGTCCTTTTTTGAA360    TTGCTGACCTTGGTTGCTTTTCTTTATTTTAGGGAGCAGGAGGTGGATTTGGTTTTATTA420    GAAGTGGGAATTGGTGGCTTACTTGACACGACCAATGTGGTAACTGGAGAGCTTGCTGTC480    ATCACCTCCATTGGGCTTGACCATCAAGAAACCTTGGGTGATAGTCTAGAAGCAATTGCA540    GAGCAGAAAGCTGGTATTTTCAAGGCTGGTAAGAAGGCAGTGATTGCGAAATTGCCTCCA600    GAAGCTAGGCTTGTCTGTCAGAAAAAAGCCGAATCTTTAGCTGTTAACCTTTATCAGGCA660    GGTCAAGATTTTTTAATGCTGAATGGTGATTTTTCAAGCTCTTTACTAAATCTTTCACAG720    CTGAACATAGGCTTAGAAGGAGTCTATCAGCAGGAGAATGCAGCCTTGGCGTTGCAAACT780    TTTCTTCTTTTTATGGGAGAAGGAAAGGAAGCTGTTGATGAACAGGCTGTAAGAAAGGCC840    TTGGAACAGACCCATTGGGCTGGTCGCTTGGAGCGTATTCGCCCACAGATTTACTTGGAT900    GGTGCTCATAACCTCCCTGCCTTGACTCGCTTGGTTGAGTTTATCAAAGAAAAAGAGCAG960    GAAGGTTATCGTCCTCAAATCCTTTTTGGAGCCTTGAAACGGAAGGATTATCAAGGGATG1020    TTAGGTTACCTGACTGAAAAATTGCCTCAGGTGGAACTCAAGGTGACCGGCTTTGACTAT1080    CAGGGGGCTTTGGACGAAAGGGATGTGACAGGTTACGATATAGTTTCTTCTTACCGAGAA1140    TTTATCAGCGATTTTGAAGAAAGAGCAGATGCTCAGGATTTGCTGTTCGTTACAGGGTCT1200    CTCTATTTTATCTCAGAAGTA1221    (2) INFORMATION FOR SEQ ID NO:4:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 407 amino acids    (B) TYPE: amino acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:    MetCysMetPheGluValGluGluCysPheHisSerArgIleGlyLeu    151015    AsnPheArgSerGlyLeuAlaGlyIleGlnGlnAlaValAspLeuLeu    202530    GlyAsnProGluGlnSerTyrProIleIleHisValThrGlyThrAsn    354045    GlyLysGlySerThrIleAlaPheMetArgGluLeuPheMetGlyHis    505560    GlyLysLysValAlaThrPheThrSerProHisIleValSerIleAsn    65707580    AspArgIleCysIleAsnGlyGlnProIleAlaAspAlaAspPheIle    859095    ArgLeuAlaAspGlnValLysGluMetGluLysThrLeuLeuGlnThr    100105110    ProAspGlnLeuSerPhePheGluLeuLeuThrLeuValAlaPheLeu    115120125    TyrPheArgGluGlnGluValAspLeuValLeuLeuGluValGlyIle    130135140    GlyGlyLeuLeuAspThrThrAsnValValThrGlyGluLeuAlaVal    145150155160    IleThrSerIleGlyLeuAspHisGlnGluThrLeuGlyAspSerLeu    165170175    GluAlaIleAlaGluGlnLysAlaGlyIlePheLysAlaGlyLysLys    180185190    AlaValIleAlaLysLeuProProGluAlaArgLeuValCysGlnLys    195200205    LysAlaGluSerLeuAlaValAsnLeuTyrGlnAlaGlyGlnAspPhe    210215220    LeuMetLeuAsnGlyAspPheSerSerSerLeuLeuAsnLeuSerGln    225230235240    LeuAsnIleGlyLeuGluGlyValTyrGlnGlnGluAsnAlaAlaLeu    245250255    AlaLeuGlnThrPheLeuLeuPheMetGlyGluGlyLysGluAlaVal    260265270    AspGluGlnAlaValArgLysAlaLeuGluGlnThrHisTrpAlaGly    275280285    ArgLeuGluArgIleArgProGlnIleTyrLeuAspGlyAlaHisAsn    290295300    LeuProAlaLeuThrArgLeuValGluPheIleLysGluLysGluGln    305310315320    GluGlyTyrArgProGlnIleLeuPheGlyAlaLeuLysArgLysAsp    325330335    TyrGlnGlyMetLeuGlyTyrLeuThrGluLysLeuProGlnValGlu    340345350    LeuLysValThrGlyPheAspTyrGlnGlyAlaLeuAspGluArgAsp    355360365    ValThrGlyTyrAspIleValSerSerTyrArgGluPheIleSerAsp    370375380    PheGluGluArgAlaAspAlaGlnAspLeuLeuPheValThrGlySer    385390395400    LeuTyrPheIleSerGluVal    405    __________________________________________________________________________

What is claimed is:
 1. An isolated polynucleotide segment encoding apolypeptide comprising the amino acid sequence of SEQ ID NO:2.
 2. Anisolated nucleic acid segment comprising a nucleotide sequence which isfully complementary to the polynucleotide segment of claim
 1. 3. Anisolated vector comprising the polynucleotide segment of claim
 1. 4. Anisolated vector comprising the nucleic acid segment of claim
 2. 5. Anisolated host cell comprising the vector of claim
 3. 6. An isolated hostcell comprising the vector of claim
 4. 7. A process for producing apolypeptide encoded by said polynucleotide segment, comprising culturingthe host cell of claim 5 under conditions sufficient for the productionof said polypeptide.
 8. An isolated polynucleotide segment comprising anucleotide sequence comprising SEQ ID NO:1.
 9. An isolated nucleicsegment comprising a nucleotide sequence which is fully complementary tothe polynucleotide segment of claim
 8. 10. An isolated vector comprisingthe polynucleotide segment of claim 8 or the nucleic acid segment ofclaim
 9. 11. An isolated host cell comprising the vector of claim 10.